Translational Regulation During Oogenesis and Early Development: the Cap-Poly(A) Tail Relationship

C. R. Biologies 328 (2005) 863–881 http://france.elsevier.com/direct/CRASS3/ Review / Revue Translational regulation during oogenesis and early development: The cap-poly(A) tail relationship

Federica Piccioni a, Vincenzo Zappavigna b, Arturo C. Verrotti a,c,∗

a CEINGE–Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Napoli, Italy b Dipartimento di Biologia Animale, Università di Modena e Reggio Emilia, Via G. Campi 213d, 41100 Modena, Italy c Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli “Federico II”, Via S. Pansini 5, 80131 Napoli, Italy Received 27 February 2005; accepted after revision 10 May 2005 Available online 8 June 2005 Presented by Stuart Edelstein

Abstract Metazoans rely on the regulated translation of select maternal mRNAs to control oocyte maturation and the initial stages of embryogenesis. These transcripts usually remain silent until their translation is temporally and spatially required during early development. Different translational regulatory mechanisms, varying from cytoplasmic polyadenylation to localization of maternal mRNAs, have evolved to assure coordinated initiation of development. A common feature of these mechanisms is that they share a few key trans-acting factors. Increasing evidence suggest that ubiquitous conserved mRNA-binding factors, including the eukaryotic translation initiation factor 4E (eIF4E) and the cytoplasmic polyadenylation element binding protein (CPEB), interact with cell-speciﬁc molecules to accomplish the correct level of translational activity necessary for normal development. Here we review how capping and polyadenylation of mRNAs modulate interaction with multiple regulatory factors, thus controlling translation during oogenesis and early development. To cite this article: F. Piccioni et al., C. R. Biologies 328 (2005).  2005 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Keywords: Capping; Polyadenylation; eIF4E; CPEB; Oocyte maturation; Translational regulatory mechanisms; Translational initiation

1. Introduction mal embryogenesis, and germ-line development [1–5]. Translational regulation of an eukaryotic mRNA is Translational control is critical for the proper regu- achieved through the orchestrated action of cis-acting lation of cell cycle, tissue induction and growth, nor- elements and trans-acting factors. Cap-dependent translation in eukaryotes requires the ordered assembly of a complex of evolutionarily conserved proteins, * Corresponding author. which starts with the binding of the translation ini- E-mail address: [email protected] (A.C. Verrotti). tiation factor 4E (eIF4E) to the 7-methyl-guanosine

(m7GpppN) cap structure at the 5 of the mRNA. Next, complexes involved in translational regulation of spe- the eIF4G factor is recruited allowing additional fac- cific mRNAs have been described and analyzed. tors (PABP, eIF4A, eIF4B, eIF1, eIF1A, eIF2, eIF3, In this review, we will focus on the molecules that among others, and the ribosomal subunits) [6] to form by binding and/or modulating the modifications occur- a complex that, after mRNA circularization, initiates ring at the end of mRNAs, the cap structure at the 5 translation [7–10]. The circularization might become and the poly(A) tail at the 3 end, are the targets of possible once an adequate poly(A) tail is present at the regulatory events that govern translation of select mR- 3-UTR [11]. NAs playing crucial roles in specific developmental eIF4E is the rate-limiting component for cap- processes. dependent translation initiation and therefore represents a major target for translational control [12,13]. eIF4E function can be regulated at different levels by a 2. Structure of eukaryotic mRNAs and variety of molecular processes. First, eIF4E transcrip- translational control tion inside a cell can be increased by growth factor stimuli [14,15]; second, the availability of eIF4E can In the nucleus, eukaryotic mRNAs are first tran- be modulated by the binding to a set of proteins that scribed as precursor mRNAs (pre-mRNAs) and sub- compete with eIF4G, a scaffold protein that aggregates sequently modified by capping, polyadenylation, and the mRNA and the ribosome [16], for eIF4E-binding, splicing. Mature mRNAs are ultimately exported into therefore inhibiting translation initiation [17];third, the cytoplasm where they can be translated into pro- elevated eIF4E activity depends upon its phosphory- teins. Capping of eukaryotic mRNAs involves the addi- lation in response to extracellular stimuli, including tion of a 7-methyl-guanosine residue at the 5 end hormones, growth factors, and mitogens, and corre- to protect this end from nuclease degradation. The lates to an increase in translation rate [16,18].The cap structure in eukaryotes can be of three types function of phosphorylated eIF4E was indeed shown m7GpppNp, m7GpppNmp, m7GpppNmpNmp (m indi- to be necessary for proper growth and development of cates a methyl group attached to the respective nu- Drosophila [19]. cleotide), and is used as a docking point for the cap- During oogenesis in many species, cytoplasmic binding protein complex that mediates the recruitment polyadenylation of a set of maternal mRNAs regu- of the small ribosomal subunit to the 5 end of the lates their translation. A general scheme implies that mRNA. elongation of a short poly(A) tail at the 3 -UTR dur- Polyadenylation occurs after cleavage of the pre- ing development is able to stimulate translation. The mRNA at the 3 end and consists of the addition of up cytoplasmic polyadenylation element binding protein to 250 adenosine residues by the poly(A) polymerase (CPEB) is a sequence-specific RNA interacting factor (PAP) enzyme. Finally, the mechanism of splicing re- that is necessary to achieve adequate poly(A) addition moves all intervening sequences from the pre-mRNA, within the cytoplasm. The rationale for the existence thus producing a mature mRNA that is competent to of a long poly(A) tail is, probably, to allow an mRNA be transported to the cytoplasm. to acquire a circular structure prior to translation ini- Once within the cytoplasm, only mRNAs that are tiation. Increasing evidence indeed suggest that the properly capped and polyadenylated are efficiently structure of an mRNA is essential for proper activity, translated. This has been demonstrated during ooge- including translation efficiency. nesis and early embryogenesis, where regulated cy- The past few years have witnessed considerable toplasmic polyadenylation of mRNAs modulate their advancements in the field of translational control dur- translation. During translation initiation, the cap struc- ing development. In particular, recent studies have re- ture is directly bound by eIF4E, and the poly(A) tail vealed the existence of various translational regula- by the poly(A) binding protein (PABP) in a manner tory mechanisms and identified in the cap-poly(A) tail that induces a synergistic enhancement of translation. interaction the primary target for multiple regulatory Translation that is both cap- and poly(A)-dependent factors. Moreover, novel repressive and stimulatory requires moreover the activity of the eukaryotic ini- F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 865 tiation translation factor 4G (eIF4G). eIF4G contains nents, the cap-binding protein eIF4E, the adaptor pro- specific binding sites for both eIF4E and PABP, thus tein eIF4G, and the poly(A)-binding protein (PABP). forming a complex that circularizes the mRNA [10]. The direct interaction of eIF4E, bound to eIF4G, to Indeed, mutations that compromise the binding of the cap structure is essential for translation initia- eIF4G with either PABP [20,21] or eIF4E [22,23] af- tion both in vivo and in vitro, while contacts between fect in vitro the synergistic stimulation of translation eIF4G and the poly(A)-bound PABP enhance transla- by the cap structure and the poly(A) tail. In this con- tion (Fig. 1A), but are not strictly required for ribo- text, the 5 cap and the 3 poly(A) tail are modifications some recruitment (see reviews [25–27]). Thus, events necessary for efficient translation. favouring the dissociation of the eIF4E–eIF4G inter- Regulatory sequences and structures within the action significantly impair cap-dependent translation mRNA modulate translation. In addition to the 5 cap and are therefore a potential means for translational and 3 poly(A) tail these include: internal ribosome control. Such a control has indeed been observed entry sites (IRESs), which direct cap-independent both in developmental processes and in tumorigene- translation initiation; upstream open reading frames sis [28,29]. (uORFs), which act as negative regulators by dimin- The functions of eIF4E and the regulation of these ishing translation from the main ORF; secondary depend on the presence on this factor of binding sur- or tertiary structures, like hairpins and pseudoknots, faces for the cap-structure of mRNAs and for vari- which often act by blocking translation initiation; and ous proteins that can modulate its activity. The three- specific binding sequences for multiple regulatory fac- dimensional structure of the eIF4E-cap complex has tors [6]. Many mRNAs are translationally controlled been solved, identifying the relevant molecular con- by sequences in their 5 and 3 untranslated regions tacts between the 5 end of mRNAs and eIF4E [30– (UTRs). Embedded within untranslated regions of eu- 35]. The cap-binding site of eIF4E was shown to be karyotic mRNAs are information specifying the way formed by a pocket, which contains two critical tryp- the RNA is to be utilized and diverse proteins bind tophan amino acid residues located close to its upper specifically to these sequences thus interpreting this and lower edges. The guanosine moiety was shown to information [24]. make contacts with these tryptophan residues. More- over, the presence of a methyl group, by introducing a positive charge, was predicted to enhance consider- 3. Eukaryotic translation initiation factor 4E ably the interaction [34,36–42]. No relevant contacts (eIF4E) and cap-binding complex assembly beyond the ones with the first nucleotide have been detected, indicating that the molecular interactions of The recruitment of the small ribosomal subunit to eIF4E with the cap are essentially identical regard- mRNA is one of the tightly regulated steps in the less of the mRNA species. Thus, since the methylated initiation of protein synthesis. Two major pathways guanosine is present on all capped mRNAs, it can be are involved in the attachment of the small ribosomal excluded that transcript-dependent efficiency of trans- units 5 to the translated region in mRNAs: the first lation is obtained by specific contacts between eIF4E depends on the 5 terminal cap structure of mRNAs, and nucleotides other than the cap. consisting of an inverted methylated guanine moiety Some evidence was presented for the existence of (m7GpppN); the second, known as cap-independent, conformational changes of eIF4E upon cap binding relies on a series of elements of complex secondary [43–45], suggesting that some or all of the factors structure, present in select mRNAs, termed IRES (in- interacting with eIF4E might be able to discriminate ternal ribosomal entry sites). The vast majority of eu- between cap-bound and apo eIF4E. One important ex- karyotic transcripts are translated in a cap-dependent ample for changes in the strength of protein–protein manner. contacts is represented by the interaction with the The cap structure represents a docking point for the inhibitors 4E-binding proteins (4E-BPs), which was cap-binding protein complex and is required for bind- shown to be significantly increased for the cap-bound ing of the small ribosomal subunit to the 5 end of form of eIF4E [46]. A further example is provided by mRNAs. This protein complex has three main compo- the interaction with eIF4G. eIF4E forms a very sta- 866 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881

Fig. 1. Modes of translational regulation during oogenesis and early development. (A)–(B). Stimulation of eukaryotic mRNA translation. (A) ‘Standard’ mRNAs. The poly(A) binding protein (PABP) binds the scaffolding protein eIF4G (4G) that in turn interacts with eIF4E (4E), thus promoting mRNA circularization and translation. (B) CPE-containing mRNAs are activated by cytoplasmic polyadenylation. The cytoplasmic polyadenylation element binding protein (CPEB) binds specific U-rich sequences named cytoplasmic polyadenylation elements (CPEs). Phosphorylated CPEB increases its affinity for the cleavage and polyadenylation specificity factor (CPSF). CPSF in turn interacts with the canonical nuclear polyadenylation signal AAUAAA and recruits poly(A) polymerase (PAP) to the 3 end of the mRNA. The consequent poly(A) tail elongation stimulates translation of dormant mRNAs. The AAUAAA sequence is written 5 to 3 for clarity. (C)–(F) Repression of eukaryotic mRNA translation. (C) ‘Standard’ mRNAs. eIF4E-binding proteins (4E-BPs) are inhibitory proteins that regulate the availability of eIF4E for interaction with eIF4G. 4E-BPs sequester eIF4E molecules that are either free or cap-bound. Hyperphosphorylation of 4E-BPs prevents interaction with eIF4E, thus allowing interaction with eIF4G and translation to start (see panel A). (D) CPEB mediates both activation and repression of translation. The repressive role of CPEB involves the interaction with Maskin. Maskin associates with the translation initiation factor eIF4E and excludes eIF4G from interacting with eIF4E, thus blocking initiation of translation of CPE-containing mRNAs. The Maskin-eIF4E complex is disrupted by cytoplasmic polyadenylation triggered by CPEB, allowing eIF4G to bind eIF4E and activate translation. Translation repression is relieved when PABP binds to the poly(A) tail and helps eIF4G displace Maskin and bind eIF4E (see panel B). (E)Bi- coid (BCD) is a specific repressor of caudal (cad) mRNA translation. The anterior determinant BCD protein acts not only as a transcriptional activator of segmentation genes, but also interacts directly with both the 3 -UTR of caudal mRNA and eIF4E to disrupt the eIF4E-eIF4G complex, thus preventing translation initiation of ubiquitously distributed cad mRNA. (F) Translational inhibition of oskar (osk) and other mRNAs. Cup is a translational regulator that acts, by interacting simultaneously with eIF4E and RNA-bound repressor molecules, in translational inhibition of specific mRNAs. Accordingly, two repressors, Bruno and Smaug, suppress translation of osk and nanos (nos) mRNAs respectively by binding to their 3 -UTRs prior to their posterior localization in the egg (osk) and embryo (nos). ble (Kd = 2–4 nM) complex with eIF4G. The eIF4G- region of eIF4E containing a Trp residue, which is binding surface within eIF4E is located distally to the also required for interaction with 4E-BPs [32,39].4E- cap-binding pocket and does not contain any known BPs are small proteins (12 Kd) showing no defined residues involved in cap binding [32,47]. NMR stud- structure in solution, which undergo conformational ies have shown that upon interaction eIF4E and eIF4G changes in the regions contacting eIF4E. All proteins mutually induce conformational changes that result known to interact with eIF4E bind to a common re- in a complex interlocking structure [30]. Binding of gion located distally to the cap-binding pocket even eIF4G to human eIF4E is mediated by the dorsal if their contacts with eIF4E, which are not yet fully F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 867 characterized, may vary considerably. The interaction interaction with eIF4E and was hence termed eIF4E- of eIF4E with the cap structure is considerably sta- binding motif. Three basic modes of action have been bilized after eIF4G binding. The RNA-binding activ- described for proteins sharing an eIF4E-binding mo- ity of eIF4G has been invoked as an explanation for tif that act antagonistically on the formation of an this. However, possible conformational changes oc- eIF4E–eIF4G complex. The first is represented by the curring upon eIF4E–eIF4G interaction that stabilize 4E-BPs, which act as general repressors of transla- cap-binding have also to be considered. A combina- tion in response to cell growth-dependent signalling tion of both mechanisms could indeed account for the events (Fig. 1C) [8,9]. The second is exemplified by enhanced stability of the eIF4E–eIF4G–PABP com- the Maskin protein, which acts in an mRNA-specific plex to the mRNA 5 end. The notable conformational manner. The target specificity of repression is obtained changes, which occur upon eIF4G-binding to eIF4E, by the interaction of Maskin with the cytoplasmic were shown to induce changes in the structure of the polyadenylation binding protein (CPEB) that in turn cap-binding site of eIF4E [30]. Binding of the PABP recognizes a uridine-rich sequence (CPE) present in protein was shown to further stabilize the cap interac- the 3 -untranslated region (3 -UTR) of selected mR- tion [45,48–51]. PABP binding to eIF4G is enhanced NAs [24,57]. The repression itself is brought about by by contacts with the poly(A) tail and RNA maximal a direct Maskin-eIF4E interaction (Fig. 1D) [58,59]. cap-binding activity likely depends upon the forma- The third mechanism is represented by the BCD pro- tion of a full eIF4E–eIF4G–PABP–poly(A) complex. tein which can both directly interact with a specific mRNA (caudal mRNA) and disrupt the eIF4E–eIF4G 3.1. Regulation of translation via competition of the complex (Fig. 1E) [60]. Recently an additional eIF4E- eIF4E–eIF4G interaction interacting protein, Cup, was identified in Drosophila and shown to play crucial roles in translational con- In addition to the 4E-BPs several other eIF4E- trol and in the localization of eIF4E during Drosophila binding proteins have been identified in a number of oogenesis and ovary development [53,55,56]. While different species, which can compete for the assem- the capability of Cup to specifically repress transla- bly of a translationally active eIF4E–eIF4G complex tion has not been experimentally verified, the findings and hence act as regulators of translation (Table 1). that Cup is able to antagonize binding of eIF4G to These are usually specialized proteins that play spe- eIF4E [54,56], and that Cup is associated to specific cific roles during developmental processes. The best mRNAs, which are transported during oogenesis and characterized examples are the X. laevis Maskin [52], whose translation is blocked during transport [53,55], the Drosophila Bicoid (BCD) and Cup proteins [53– would suggest that Cup negatively controls translation 56]. These proteins share with eIF4G and the 4E- as well (Fig. 1F). Cup has been suggested to act via BPs the characteristic motif Tyr–X–X–X–X–Leu– a mechanism similar to that of Maskin [61]. There (where X represents any residue and is leucine, is evidence that Cup is recruited to the oskar (osk) methionine or phenylalanine), which is required for and nanos (nos) mRNAs by the Bruno and Smaug proteins, respectively, which specifically contact these mRNAs (Fig. 1F) [53–55]. However, Cup might play a Table 1 role in osk translational repression also independently Putative functions of eIF4E interactors in translation of its binding to osk mRNA since the loss of Cup activ- Interactors Function of the interactions in translation ity was shown to have a more pronounced effect on osk eIF4G’s Stimulation of translation of all mRNAs translation than preventing Bruno from binding to osk Maskin Repression of translation of CPE-containing mRNAs mRNA. Moreover, Cup was shown to be associated Bicoid Repression of translation of caudal mRNA Cup Repression of translation of oskar and other mRNAs with Barentz, a protein required for the localization 4E-BP’s Repression of translation of all mRNAs of the osk mRNA, indicating that Cup plays a role in Emx2 Local control of translation in olfactory sensory the localization of the osk mRNA within the oocyte as neurons well [55]. Interestingly, the localization and the over- HOXA9 Stimulation of the nuclear export of cyclinD1 and all amount of the eIF4E protein, which accumulates at ODC mRNAs the posterior end of the developing Drosophila oocyte, 868 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 arealteredinacup mutant background [55,56],fur- plexes for cap-binding would determine the levels of ther suggesting that Cup plays more general functions translational activity within cells. in translational regulation within the oocyte, likely by Experimental variations of eIF4E levels in vivo controlling the translation of a number of different have proven to be valuable in estimating the stabil- mRNAs. Indeed, mutation of cup was shown to have ity of the translation initiation complex within cells. a substantial effect on oocyte maturation and ovary The overexpression of eIF4E by up to a factor 100 development [62,63], and a reduction of eIF4E lev- in yeast has only minor effects on growth rates [72], els within ovaries causes a significant aggravation of while a modest increase in translation rates could be the cup mutant phenotype [56]. Thus, the role of Cup observed in X. laevis cells [73]. Thus the overall trans- might be to coordinate localization of a number of mR- lation levels are apparently not significantly affected NAs during Drosophila oogenesis and to assure their by increases in eIF4E availability. Along these lines, translational repression during transport. These tran- a 30% reduction of eIF4E wild-type levels is toler- scripts might encode products asymmetrically distrib- ated in yeast without apparent effects on growth rate; uted within the oocyte to assure its proper maturation. and translation in yeast is seemingly affected only as soon as eIF4E levels fall close to or below those of 3.2. eIF4E levels and translation total mRNA [45]. Thus, at least under conditions of high translational activity, eIF4E levels do not seem to A crucial aspect of the regulation of translation ini- influence bulk translation. Nevertheless, alterations of tiation is the relationship between eIF4E levels and eIF4E levels that are supposed to leave bulk translation translational activity within cells. Changes in the abun- unchanged can have profound effects on the physi- dance or activity of eIF4E have been reported in sev- ology of cells. For example, in yeast, reductions in eral cases and linked to tumorigenesis [29], adapta- eIF4E levels that do not impair general translation, sig- tion to environmental stresses [64], and developmental nificantly affect cell morphology, ribosome biogenesis processes [28]. The relative amount of eIF4E and other and cell cycle progression [45,74]. The importance of translation initiation factors has been a controversial eIF4E levels for the translation of select mRNAs has issue. It has been initially reported that eIF4E levels been recently shown in the case of the Drosophila are limiting, at least in mammalian cells and reticu- neuromuscular junctions (NMJ). eIF4E accumulates locyte lysates [65,66]. However, more recent data in- at Drosophila larval NMJs to sustain local transla- dicate that eIF4E is present in reticulocyte lysates in tion of specific mRNAs required for synaptic function. excess over eIF4G, and that in yeast it is equimolar The product of the pumilio (pum) gene was shown to to ribosomes and to other initiation factors. Thus, ap- repress local eIF4E accumulation at synapses of the parently, in reticulocytes, yeast, and D. melanogaster NMJs by binding directly to the mRNA of eIF4E.Lo- cells, it is the availability of eIF4G, rather than that of cal eIF4E levels were found to be relevant for the mod- eIF4E, which limits the frequency of translation ini- ulation of the translation efficiency of the glutamate tiation [34,45,67,68]. The availability of eIF4E could receptor GluRIIA mRNA and thus for synaptic trans- be modulated by the activity and/or abundance of 4E- mission [75]. Furthermore, overexpression of eIF4E BPs within cells. Under active growth conditions the can cause malignant transformation of cells, an effect 4E-BPs are in a hyperphosphorylated state, due to that can be reversed by reducing eIF4E levels. Accord- the activity of the FRAP/mTOR kinase [69], thus in- ingly, elevated levels of eIF4E are observed in cancer hibiting binding to eIF4E [70,71]. In these conditions, cells, correlating with the severity of the disease [29]. eIF4E is likely present predominantly as a complex Another critical parameter for the modulation of with eIF4G or in the apo eIF4E form. When cells are translational efficiency within cells is the stability not actively growing and hence the levels of protein of the cap binding complex-mRNA interaction. In synthesis are reduced, the 4E-BPs remain in a dephos- vitro experiments using recombinant eIF4E, eIF4G phorylated state and thus are able to bind eIF4E, thus and PABP indicate that cap binding complexes are reducing the availability of free eIF4E. The extent of stable and dissociate relatively slowly from mRNAs. 4E-BPs phosphorylation and the efficiency of compe- However, in cap-binding assays using purified human tition between eIF4E–4E-BP and eIF4E–eIF4G com- proteins, the addition of eIF4B, a factor with heli- F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 869 case activity, can destabilize preassembled cap com- transforming cells as efficiently as the wild-type pro- plexes, leading to an accelerated turnover of cap bind- tein, suggesting that the oncogenic capacity of the cap- ing complex-mRNA interaction [76]. This suggests binding protein may be based on the control of mRNA that in vivo as opposed to in vitro eIF4B and possibly export rather than on translational control [43]. These other factors may alter the stability of the cap complex. results would furthermore suggest that a pool of free In conclusion, changes in the concentration of eIF4E might function in the export of specific mRNAs eIF4E apparently produce two distinct types of re- without significantly competing with eIF4E, bound to sponse, one relating to general translation, which is eIF4G, for binding to the cap structure. Two human at best subtle, and the other on specific aspects of proteins have been identified as additional interactors cellular function, which can be in several cases consid- of eIF4E, which might function as negative regulators erable. A possible explanation for this discrepancy is of eIF4E-dependent nuclear export of specific mR- that a specific subset of mRNAs within cells has a spe- NAs: the PML (promyelocitic leukaemia) and PRH cial requirement with respect to eIF4E function and is (proline-rich homeodomain protein) proteins [43,82]. thus particularly sensitive to alterations in the level of The interactions between these proteins and eIF4E re- functional eIF4E. Examples of these mRNAs are the quire the dorsal Trp residue of eIF4E as do the interac- yeast CLN3 transcript [74], and the VPF [77], FGF-2 tions with eIF4G and the 4E-BPs. However, PML and [78], CyclinD1 [15], ODC [79], and Pim-1 [80] mam- PRH do not contain the conserved eIF4E binding mo- malian mRNAs. The regulation of these transcripts tif, although PRH contains a related sequence in which via eIF4E has been suggested to involve two possi- the hydrophobic residue () is exchanged for a gluta- ble different mechanisms. The first mechanism might mine [82]. While 4E-BPs and eIF4G stabilize binding be based on subtle differences in the affinity of the of eIF4E to the cap structure, both PML and PRH re- cap-binding complex for different mRNAs. Although, duce the affinity of eIF4E for the cap structure. The as discussed above, only minor differences have been negative effect on eIF4E’s cap binding activity is prob- pointed out in the capability of the cap-binding com- ably linked to the regulatory role of PML and PRH on plex to discriminate between different mRNA species, the export activity of eIF4E. these differences could become critical in the presence Interestingly, in addition to PRH and BCD other of limiting amounts of eIF4E within cells. The second homeodomain transcription factors have been recently one could involve 5-UTR secondary structures and reported to be able to directly interact with eIF4E. the consequent differential dependency on the heli- Indeed, among the known homeodomain-containing case activity of eIF4A by different mRNAs to bind the proteins about 200 were found to contain a puta- small ribosomal subunit. Thus the limitation in the as- tive eIF4E binding sequence [82]. One of these, the sembly of a cap complex might limit the resolution of HOXA9 homeodomain protein, was shown to func- secondary structures within a given mRNA and hence tionally interact with eIF4E. HOXA9, however, un- affect its translation. like PRH, appears to be a stimulator of eIF4E activity. HOXA9 can antagonize the interaction of PRH 3.3. eIF4E and nuclear export of mRNAs with eIF4E thereby modulating the PRH-mediated repressive action on eIF4E activity. HOXA9 apparently Another aspect of transcript specific regulation of modulates both the nuclear as well as the cytoplasmic translation is the dependence of select mRNAs on functions of eIF4E. In the nucleus, HOXA9 was found eIF4E for their nuclear export. Despite the fact that the to promote the eIF4E-dependent nuclear export of the eIF4E-dependent mRNA export is still poorly charac- cyclinD1 and ornitine decarboxylase (ODC)mRNAs, terized, the human cyclinD1 message and other mR- whereas in the cytoplasm it was found to stimulate NAs have been shown to be exported via an eIF4E- the efficiency of translation of the ODC mRNA. [83]. dependent mechanism [81,82]. Thus, protein synthesis Similarly, the Emx2 homeodomain transcription factor for some mRNAs might be sensitive to eIF4E levels was found to be associated with eIF4E in the olfac- independently of translational control. Interestingly, a tory sensory neuron axons, suggesting that it may play mutant of eIF4E that cannot function in translation a role in controlling the local translation of mRNAs but can mediate cyclinD1 mRNA export, is capable of encoding proteins mediating synaptic plasticity and/or 870 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 axon guidance [84] (Table 1). It seems likely that additional homeodomain protein–eIF4E interactions will be reported in the future reinforcing the knowledge that control of translation via eIF4E may be directly linked to the activity of factors controlling patterning and regional identities in development.

3.4. eIF4E and mRNA turnover

Several pathways of mRNA degradation require removal of the cap structure since the presence of the cap inhibits the activity of 5 → 3 exonucleases that are crucial for this process (Fig. 2). Specific decapping enzymes have been found in several organisms; the best characterized are the yeast Dcp1 and Dcp2 (reviewed in [85]). Dcp cleavage activity on the cap requires access by these enzymes to parts of the cap structure that are engaged in eIF4E binding. Evidence both in vitro [86,87] and in vivo [86,88] for a competition between eIF4E and Dcp activity have been provided. It can be thus speculated that a decrease in eIF4E and eIF4G activity could lead to mRNA degradation, and more generally cap-binding complex destabilization could correspond to an acceleration of mRNA degradation. In yeast, the shortening of the poly(A) tail to about ten nucleotides is a prerequisite for decapping Fig. 2. Schematic representation of eukaryotic mRNA turnover. Sev- and subsequent mRNA degradation. Since the interac- eral pathways of mRNA degradation require removal of the cap tion of the yeast poly(A) binding protein (Pab1) with structure since the presence of the cap inhibits 5 → 3 exonucle- the poly(A) tail was shown to require a larger num- ase activities that are crucial for this process. The first step is the ber of nucleotides [89], it could be hypothesized that shortening of the poly(A) tail to about ten nucleotides, a prerequisite for removal of cap structure by Dcp enzymes. Following decap- poly(A) tail shortening could lead to PABP dissocia- ping, mRNA is degraded by 5 –3 exonucleases. Another pathway tion and the consequent destabilization of the eIF4G– of mRNA degradation independent of decapping is not represented eIF4E-cap interaction, rendering the cap accessible for here. Dcp activity. However, yeast strains that carry a mutation in Pab1, able to bind poly(A) but not eIF4G, mRNA stability could be influenced by the competi- show normal deadenylation and decapping activities tion for cap access between the aforementioned decap- [90], indicating that the correlation between poly(A) ping accessory factors and eIF4E. According to this tail length and mRNA turnover in cells is less obvi- view, shortening of the poly(A) tail would induce asso- ous than predicted. Removal of the cap structure re- ciation of the Lsm1–7 complex to the mRNA, leading quires in addition the activity of a number of accessory to the dissociation of the cap-binding complex via the factors such as the enhancers of decapping Edc1 and helicase activity of Dhh1 and the consequent gain of Edc2, the mRNA-binding protein Pat1, the Lsm1–7 access of Dcps to the cap [93]. proteins, which bind Dcp proteins and are recruited to the mRNA after deadenylation, and the RNA he- 3.5. eIF4E phosphorylation licase Dhh1 [91–93]. These proteins were shown to associate with mRNAs forming a large complex after In addition to its association with inhibitory eIF4E- deadenylation but before decapping [93]. Thus rather binding proteins, eIF4E is regulated through phospho- than by a direct competition between Dcps and eIF4E, rylation [16]. The MAP kinase-interacting kinases 1 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 871 and 2 (Mnk1 and Mnk2), which are phosphorylated lation rate [16]. Recently, genetic data in Drosophila and activated by the mitogen-activated protein ki- demonstrated that eIF4E phosphorylation is biologi- nases (MAPKs) ERK and p38, phosphorylate eIF4E cally significant and necessary for normal growth and on Ser209 [94], the major site of phosphorylation of development, because a point mutation of the known eIF4E in mammals [95–97]. In mice lacking both the eIF4E phosphorylation site (Ser 251, corresponding to Mnk1 and Mnk2 genes, eIF4E is indeed not detectably Ser 209 of mammalian eIF4E) causes a delay in devel- phosphorylated at Ser209 [98]. However, these mice opment and reduction in body size [19]. are viable, fertile, develop normally, and show neither In Drosophila, the Lk6 kinase, the closest homo- general protein synthesis nor cap-dependent transla- logue of mammalian Mnk kinases, is responsible for tion defects, suggesting that eIF4E phosphorylation at eIF4E phosphorylation [103].Lossoflk6 function the conserved Ser209 is not essential for cell growth leads to slower development, and reduced viability during development [98]. Surprisingly, Mnk1 does and adult size, demonstrating that Lk6 function is re- not interact with eIF4E directly, but binds to the C- quired for organism growth. Moreover, in lk6 mu- terminal region of eIF4G. Thus, it has been proposed tant flies eIF4E phosphorylation is dramatically re- that eIF4G provides a docking site to bring Mnk1 next duced [104]. Double Mnk1/Mnk2 knockout mice have to its substrate eIF4E [99]. While Mnk1 appears to be demonstrated that both kinases are dispensable for de- the major physiological eIF4E kinase [16], protein ki- velopment and growth. These results differ from the nase C (PKC) has been shown to phosphorylate eIF4E one obtained in Drosophila probably because either as well [97], and a PKC consensus sequence does in- redundancies or compensatory effects occur in mam- deed surround Ser-209 [96]. mals [104]. In addition other factors may influence The phosphorylated form of eIF4E binds to mRNA the phosphorylation status of eIF4E. The translational caps 3–4-fold more tightly than the non-phosphorylat- regulator Cup, which interacts with eIF4E to modu- ed form [37]. However, recent findings have demon- late development and growth of the Drosophila ovary, strated that phosphorylation of eIF4E impairs its abil- likely plays a role in the control of the phosphoryla- ity to bind capped mRNA [40,100] and increases the tion status of eIF4E within the developing ovary, since rate of dissociation of eIF4E from the cap analogues in cup mutant ovaries the amount of phosphorylated or capped RNA [40], suggesting that phosphorylation eIF4E appears to be reduced [56]. occurs only after mRNA binding. Thus it is a dephos- In the marine mollusc Aplysia californica, eIF4E is phorylated eIF4E that binds an mRNA cap, leading to phosphorylated at Ser207 (corresponding to Ser 209 the formation of the translation initiation complex and of mammalian eIF4E) by protein kinase C [105].An recruitment of the ribosome. antibody that specifically recognizes the phosphory- Scheper and Proud [101] proposed two possi- lated form of Aplysia eIF4E showed that the level of ble mechanisms related to eIF4E phosphorylation: phosphorylated eIF4E correlates with the basal rate (A) phosphorylation occurs immediately after the as- of translation in the nervous system [106]. More- sembly of the initiation complex and facilitates eIF4E over, in this organism eIF4E dephosphorylation can release from the cap structure, allowing the ribosome trigger a switch to IRES-mediated translation [107]. to begin scanning; (B) phosphorylation occurs later in eIF4E phosphorylation might be involved in synap- the initiation process and enhances the release of ini- tic plasticity and memory as well. In murine hip- tiation factors from the cap-structure, rendering the pocampal neurons, reduction of ERK activity led to a cap-binding factors available for the translation of decrease of neuronal activity-induced translation and other mRNAs. Thus, phosphorylation could play an phosphorylation of eIF4E and other translation fac- important role in ‘reprogramming’ of the translational tors [108]. machinery. Phosphorylation of eIF4E is enhanced by a variety of agents that stimulate translation, including insulin, 4. CPEB and cytoplasmic poly(A) addition hormones, growth factors, and mitogens [95,96,102]. Thus, it has been proposed that eIF4E phosphorylation The initial polyadenylation of an mRNA molecule plays a positive role in cell growth by enhancing trans- occurs in the nucleus. Whereas the 7-methylguanosine 872 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881

the 3 end [109]. These modifications confer mRNA stability, promote translation efficiency, and have a role in the transport of processed mRNA from the nucleus to the cytoplasm [110]. Once mRNAs emerge from the nucleus, the des- tiny of their poly(A) tails can vary: they are slowly shortened in most cells [111] thus gradually diminish- ing their translation rate, or undergo dramatic changes thus regulating translation at specific times and places in early development. During oocyte maturation and early embryogenesis, specific mRNAs destined to be stored in a translationally dormant state are deadenylated rapidly, causing their repression [112]. Later, the same mRNAs receive a long poly(A) tail and become translationally active [111]. Oocytes and early embryos are transcriptionally inactive and require rapid changes in the proteins they contain to regulate development [111]. A form of translation regulation is the poly(A) tail lengthening of specific mRNAs, a process Fig. 3. Mechanism of polyadenylation in the nucleus. The poly(A) called cytoplasmic polyadenylation and driven by cy- tail is added by the poly(A) polymerase (PAP) after transcription has ended. Nuclear poly(A) addition requires a series of cis-acting toplasmic enzymes. sequences and specific trans-acting proteins bound to them. The nuclear polyadenylation signal AAUAAA, bound by the cleavage 4.1. Translational regulation through cytoplasmic and polyadenylation specificity factor (CPSF), is positioned 10–30 polyadenylation nucleotides upstream of the polyadenylation site (represented by a 5 -CA-3 di-nucleotide) that is followed, after 10–20 nucleotides, by a G/U-rich region bound by the cleavage stimulation factor Cytoplasmic polyadenylation is generally corre- (CstF). After binding of these factors, the emerging RNA molecule lated with translational activation and deadenylation is cleaved, just downstream of the CA sequence, and the poly(A) with translational repression. Translational control by polymerase adds 100–250 adenosine residues to the 3 end. cytoplasmic polyadenylation is necessary for mouse and Xenopus oocyte maturation as well as Drosophila embryogenesis [111,113–115]. Induction of translation of many maternal mRNAs, such as c-mos and cap structure is added at the 5 end as soon as tran- tissue plasminogen activator (tPA) in vertebrates [112, scription has started, the 3 poly(A) tail is added by 113,115], and bicoid, Toll, torso, and hunchback in the poly(A) polymerase (PAP) after transcription has Drosophila [116,117] is accompanied by their cyto- ended. Nuclear poly(A) addition requires a series of plasmic polyadenylation and plays an essential role in cis-acting sequences and specific trans-acting proteins the early development of these organisms. The cyto- bound to them (Fig. 3). The nuclear polyadenyla- plasmic polyadenylation reaction, at least in Xenopus tion signal AAUAAA, bound by the cleavage and and mouse, is regulated by sequences in the 3 UTR polyadenylation specificity factor (CPSF), is posi- of mRNA that include the hexanucleotide AAUAAA tioned 10–30 nucleotides upstream of the polyadeny- (a highly conserved sequence required also for nu- lation site (represented by a 5 -CA-3 di-nucleotide) clear pre-mRNA cleavage and polyadenylation) and that is followed, after 10–20 nucleotides, by a G/U- U-rich sequences (e.g., UUUUUAU) called cytoplas- rich region bound by the cleavage stimulation factor mic polyadenylation elements (CPEs). CPEs activity (CstF). After binding of these factors, the emerging can be modulated also by other sequences located RNA molecule is cleaved, just downstream of the CA in the 3-UTR [118–120]. Insertion of a CPE into sequence, and the poly(A) polymerase adds 100–250 the 3UTR of a reporter mRNA causes polyadenyla- adenosine residues, according to the animal species, to tion and translational induction during oocyte matu- F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 873 ration or early development [112,118,119,121].How- mRNA [129]. Moreover, phosphorylation of CPEB in- ever, not all mRNAs that contain CPEs undergo cy- creases its affinity for CPSF [129], which in turn inter- toplasmic polyadenylation, suggesting that this se- acts to the AAUAAA sequence and recruits poly(A) quence alone is not sufficient to induce polyadenyla- polymerase to the 3 end of the mRNA (Fig. 1B) [123, tion [114]. At least three factors are involved in cy- 130]. toplasmic polyadenylation: the poly(A) polymerase, The mechanism of polyadenylation-induced trans- similar to nuclear poly(A) polymerases in mammalian lation is still unclear; it has been proposed that in re- cells [122], CPSF [123], also known to be essential sponse to elongation of the poly(A) tail some mRNAs in nuclear polyadenylation, and CPE-binding protein undergo a 5 cap-specific 2-O-methylation that en- (CPEB). hances translation efficiency during oocyte maturation CPEB, a 62-kDa protein with two RNA recogni- [131]. A second mechanism may involve the poly(A) tion motifs (RRMs) [124] and a zinc finger domain binding protein (PABP). Long poly(A) tails are bound [125], was first described as a protein critical for efficiently, every 20–30 residues, by PABP that is able cytoplasmic polyadenlytion in Xenopus oocytes. Im- to bind also eIF4G. This has led to the idea that the as- munodepletion of the protein from Xenopus egg extracts renders them incapable of in vitro polyadenyla- sociation of PABP with mRNAs bearing long poly(A) tion, while partial polyadenylation activity is restored tails can help to recruit eIF4G for binding with eIF4E by supplementing the depleted extract with in vitro thus promoting translation [93,132]. PABP contributes synthesized CPEB [124]. CPEB is an RNA-binding to translation in Xenopus oocytes and a mutant form of protein that binds specifically to the CPEs and con- eIF4G, unable to interact with PABP, reduces transla- trols the polyadenylation of several mRNAs, including tion of polyadenylated mRNAs and inhibits matura- those encoding c-Mos, several cyclins, and cdk2 dur- tion of oocytes [133]. ing oocyte maturation in Xenopus.InXenopus,c-mos CPEB has been described as a protein able to mRNA encodes for a serine/threonine kinase which activate translationally dormant mRNAs in Xenopus regulates oocyte maturation [126]. Removal of two oocytes by elongation of their poly(A) tails [24]. cis-acting sequences contained within the 3 -UTR of CPEB, however, does not act only in activation of endogenous c-mos mRNA results in the complete in- translation, since it has also been shown to repress hibition of oocyte maturation [115]. Polyadenylation translation of mRNAs bearing a CPE in the 3-UTR of c-mos mRNA is also required for its translational [134]. Thus, CPEB, which appears to be constantly activation in mice and this process is dependent on bound to specific cis-acting sequences in the 3 -UTR the presence of two CPEs in the 3 -UTR [113].In- of mRNA, mediates both repression and activation of jection of CPEB antibody into oocytes blocks their translation and these opposing behaviours may be ex- progesterone-induced maturation due to inhibition of plained by its diverse interactions. The repressive role cytoplasmic polyadenylation and translation of c-mos of CPEB involves the interaction with Maskin, which mRNA, thus suggesting that CPEB is critical for early was isolated as a CPEB-binding protein. Maskin con- development [127]. Male and female CPEB gene null tains an eIF4E-binding motif [16,52] and bridges mice are viable and develop normally, but are in- eIF4E with CPEB thus preventing the binding of fertile [128]. In addition, oocytes of CPEB knockout animals fail to polyadenylate and translate CPE- eIF4G, required to correctly position the 43S ribo- containing mRNAs [128]. somal subunit on the mRNA, to eIF4E and blocking The initiation of cytoplasmic polyadenylation re- initiation of translation of CPE-containing mRNAs quires the activity of the Eg2 kinase, a member of Au- (Fig. 1D) [135]. The Maskin-eIF4E complex is dis- rora family of serine/threonine protein kinases. CPEB rupted by cytoplasmic polyadenylation triggered by is phosphorylated on Ser174 in vitro, and immunode- CPEB phosphorylation [136], allowing eIF4G to bind pletion of Eg2 from oocyte extracts prevents CPEB eIF4E and activate translation [52]. Translation re- phosphorylation on this amino acid residue. CPEB pression is relieved when PABP binds to the poly(A) phosphorylation is both necessary and sufficient to tail and helps eIF4G displace Maskin and bind eIF4E stimulate polyadenylation and translation of c-mos (Fig. 1B) [52]. 874 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881

Table 2 CPEB-like molecules and their functions in development mRNA targets Function in development xCPEB (Xenopus laevis) c-mos Oocyte maturation Cdk2 Oocyte maturation cyclin B1 Oocyte maturation p82 (Spisula solidissima) cyclin A Oocyte maturation Orb (D. melanogaster) oskar Posterior determinant mCPEB (Mus musculus)c-mos Oogenesis/Spermatogenesis CPB-1 (C. elegans) N/A Spermatogenesis FOG-1 (C. elegans) N/A Spermatogenesis hCPEB (H. sapiens)N/AN/A

4.2. CPEB, CPEB-like molecules, and transcript of localized transcripts [149]. Multiple cis-acting el- localization during early development ements within the 3-UTR of orb appear indeed to be required for directing its own localization during CPEB homologues have been identified in humans Drosophila oogenesis [139]. (hCPEB) [137], clams (p82) [138],flies(Orb)[139] Localization-dependent translation is a common and zebrafish (Zorba) [140]. In addition, four iso- translational regulatory mechanism in development forms have been cloned in mice (mCPEB-1, mCPEB- [150]. In this context, mRNAs can be localized at 2, mCPEB-3, mCPEB-4) [141,142] and four CPEB specific sites within the cell where they are trans- homologues have been identified in Caenorhabditis lated. oskar mRNA is localized to the posterior pole elegans (CPB-1, CPB-2, CPB-3, and FOG-1) [143, of the oocyte where Oskar protein, a posterior deter- 144]. In worms, two CPEB homologues, CPB-1 and minant, directs posterior cell fates [151,152]. Transla- FOG-1, have key functions in spermatogenesis and tion of oskar mRNA is repressed before the localiza- are dispensable for oogenesis [144], whereas in frogs, tion process commences. An ovarian protein, named flies, and clams, CPEBs are essential during oogenesis Bruno, has been implicated in this translational re- [120,139,145] (Table 2). pression. The repressor protein Bruno binds specifi- In Drosophila, translational activation of bicoid cally to sequences called BRE (Bruno Response Ele- (bcd) mRNA is required for determination of ante- ments) present in the 3UTR of oskar mRNA [153]. rior structures in the embryo. The poly(A) tail of the Moreover, it has been shown that a germ-line pro- bcd mRNA is extended after fertilization and bcd tein, named Cup (see eIF4E section), acts together is translated at that time. A long poly(A) tail is required and might be sufficient for translation of bcd with Bruno to repress oskar translation (Fig. 1F) [116]. For certain mRNAs, however, the extension [53]. Bruno shares a 50% sequence identity with process, instead of poly(A) tail length per se, seems the Xenopus deadenylation promoting factor EDEN- to be necessary to activate translation [119,146].In BP [154], EDEN-dependent deadenylation promotes Drosophila, no cis-acting elements involved in cyto- rapid poly(A) tail shortening of a set of maternal plasmic polyadenylation have been identified so far. mRNAs after fertilization of Xenopus oocytes and However, CPEB is 62% identical to Orb [124],an represents likely a conserved mechanism to inhibit oocyte-specific RNA-binding protein involved in RNA translation in metazoa [154]. localization and in antero-posterior and dorso-ventral Posterior localization of oskar mRNA is essential patterning during Drosophila oogenesis [139,147]. for its translation. oskar mRNA is transported to the The loss of orb activity in Drosophila oocytes blocks posterior pole of the oocyte, where Orb protein is al- the polyadenylation and translation of certain maternal ready localized, by the RNA-binding protein Staufen mRNAs [139,147,148].TheDrosophila CPEB ho- [155,156]. Orb then activates translation of osk by molog Orb binds directly to oskar mRNA 3UTR and inducing its cytoplasmic polyadenylation, since os- activates oskar translation [148]. mRNA localization kar mRNA in orb mutant flies has a shorter poly(A) appears mediated by sequences located in the 3UTR tail than in wild type [148]. Orb mutants localize os- F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 875 kar transcripts with a shortened poly(A) tail that fails plasmic polyadenylation and translational activation. to enhance oskar translation, thus resulting in poste- This element was originally identified in the 3-UTR rior patterning defects [157]. In contrast, it has been of Mos mRNA and shown to function during Xenopus proposed that translation of oskar mRNA could be oocyte maturation [166]. poly(A) independent [158,159] leading to the idea that polyadenylation is not the determining event of translation derepression. Cytoplasmic polyadenylation 5. Conclusion and future challenges might be important for enhanced and efficient translation [157]. Multiprotein complexes regulate translation initia- A growing body of experimental evidence demon- tion of eukaryotic mRNAs by modulating the cross- strates that CPEB is expressed also in neurons [160, talk between the cap structure and the poly (A) tail. 161]. The protein translation machinery is present in The 5 and 3 termini of mRNAs represent therefore dendritic processes and it has been proposed that spe- major targets for translational control during develop- cific mRNAs may be translationally repressed before ment and pathological conditions such as cancer. they reach the dendrites where repression is relieved The binding of eIF4E to the cap structure is mod- [161,162]. CPEB, controlling translation of specific ulated by its phosphorylation. It is not yet clear, how- mRNAs located in dendritic processes, may promote ever, whether phosphorylation increases or diminishes synaptic plasticity within dendrites [161,162].CPEB the binding affinity to the 7-methyl-guanosine and re- may be activated following synaptic stimulation and mains to be ascertained if multiple phosphorylation promote the polyadenylation and translation of CPE- sites are involved in this process. Genetic and bio- containing mRNAs in dendrites [58,161,163].CPEB chemical data indeed suggest that phosphorylation of facilitates mRNA translation as well as transport [161, the evolutionary conserved Ser209 (human coordi- 163]. CPEB also colocalizes with Maskin in CPE- nates) alone does not explain all its effects in vivo. containing RNA particles that are transported along Proteomic techniques coupled to genetic analysis, microtubules to dendrites [163]. Overexpression of obtained in a simple animal model system such as CPEB enhances RNA transport, whereas overexpres- Drosophila, could unravel the modality and role of sion of a CPEB mutant protein, which is unable to phosphorylation during eukaryotic translation initia- associate with kinesin and dynein, inhibits transport tion and early development. [163].InAplysia it has been identified a neuron- CPEB-dependent translational regulation promotes specific isoform of CPEB that regulates the synap- germ-line development in diverse organisms, varying tic protein synthesis in an activity-dependent manner from flies to mammals. Mental deficits in humans are [164]. often linked to sterility, thus suggesting a role of CPEB in learning and memory activities. Moreover, the ex- 4.3. CPE- and CPEB-independent cytoplasmic istence of multiple CPEB isoforms in mammals may polyadenylation imply tissue-specific translational regulatory mechanisms. A multi-organisms approach will help unravel The mechanism of translational regulation by cy- the inhibitory/stimulatory mechanisms of CPEB and toplasmic polyadenylation appears to be tightly cou- its multiple targets. pled to the binding of CPEB to CPE sequences. In In recent years data from many laboratories have progesterone-stimulated Xenopus oocytes, however, uncovered the general mechanisms of protein syn- early cytoplasmic polyadenylation, and subsequent thesis. Novel mRNA-specific translational regulatory translational activation, of a class of maternal mR- mechanisms continue however to be discovered and NAs occurs independently of a CPE and CPEB [165]. the number of factors involved in such regulations is On the contrary, late cytoplasmic polyadenylation oc- increasing proportionally. In addition, several distinct curs only via CPE- and CPEB-dependent mechanisms. eIF4E isoforms, with specific developmental expres- A novel type of sequences contained within the 3- sion profiles, have been identified in Drosophila [167] UTR of early mRNAs, named polyadenylation re- and a similar scenario could be soon unravelled for sponse elements (PREs), temporally direct early cyto- members of the CPEB family. It is therefore expected 876 F. Piccioni et al. / C. R. Biologies 328 (2005) 863–881 that more examples of regulative protein interactions [12] S. Mader, N. Sonenberg, Cap binding complexes and cellular will be reported in the near future, providing support growth control, Biochimie 77 (1–2) (1995) 40–44. to the idea that eIF4E and CPEB functions can be reg- [13] L. McKendrick, V.M. Pain, S.J. Morley, Translation initiation factor 4E, Int. J. Biochem. Cell Biol. 31 (1) (1999) 31–35. ulated by the interaction with a wide variety of tissue [14] R.M. Jones, J. Branda, K.A. 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